17alpha, 21-dihydroxypregnene esters as antiandrogenic agents

ABSTRACT

17α,21-Dihydroxypregna-4,9-diene-3,20-dione and 17α,21-dihydroxypregna-4-ene-3.20-dione 17 and/or 21 esters of having remarkable antiandrogenic activity, and the processes for the preparation thereof.

The present invention relates to 17α,21-dihydroxypregnene esters,processes for the preparation thereof and the use thereof asantiandrogenic agents.

PRIOR ART

A number of corticosteroids have been used as anti-inflammatory,anti-rheumatic, anti-allergic and anti-shock agents.

In particular, 11-deoxy-hydrocortisone esters and derivatives thereofhave been widely used as anti-inflammatories.

No 17α,21-dihydroxypregnene mixed esters are known, while 17 and 21 acylderivatives with equal aliphatic chains having no more than four carbonatoms have been described.

Carboxylic acids 17 or 21 monoesters having no more than six carbonatoms are also known.

For example, the preparation of 17α,21-diacetoxypregna-4-ene-3,20-dioneis disclosed in U.S. Pat. No. 3,530,038, that also mentions the use ofpropionyl derivatives and a series of aliphatic acyl derivatives havingup to six carbon atoms chains.

U.S. Pat. No. 3,152,154 discloses the preparation of21-hydroxypregna-4,9-diene-3,20-dione-17α-butyrate, which is used as anintermediate for the preparation of3,21-diacyloxy-17α-butanoyloxypregna-3,5,9-triene-20-one wherein the 3and 21 acyls are the same and are acetyl, propanoyl, butanoyl andisobutanoyl. All of the examples cited in these documents concerncompounds wherein the 17α and 21 positions are esterified with the sameacyl group.

The preparation of 21-acetoxypregna-4-ene-3,20-dione-17α-dimethylpropionate is described in Liebigs Ann. Chem. 1983, 705-711 as the onlyexample of mixed esters: the Authors state that the preparation of themixed ester is possible only when the substituent in position 21 is anacetyl.

U.S. Pat. No. 3,530,038 discloses a process for the preparation of11β-17α-21-trihydroxy steroids which comprises subjecting11-deoxy-17α-OR-21-OR′ steroids, wherein R is a carboxylic acid residueof 1-18 carbon atoms and R′ is hydrogen or an acyl of 1 to 18 carbonatoms, to microbiological oxidation with Curvularia for obtaining thecorresponding 11β-hydroxy steroid.

According to the same patent, compounds of the pregnane, androstane orestrane series are mentioned as possible starting steroids, but nomention is made of any transformation of 11-deoxy-17α-OR-21-OR′ steroidswherein R is an acyl of 1-18 carbon atoms and R′ is hydrogen.

The preparation of these products was described by R. B. Turner (J. Am.Chem. Soc. 75 (1953) 3489) with reference to the preparation ofpregna-4-ene-3,20-dione-17α,21-diacetate and by R. Gardi et al. (Gazz.Chim. It. 93 (1963) 431-450).

Finally, U.S. Pat. No. 3,780,177 discloses the preparation of21-hydroxy-pregna-4,9-diene-3,20-dione-17α-butanoate by means oforthobutyrates and the use thereof as an intermediate for thepreparation of 6α,9α-difluoroprednisolone 17-butanoate-21 esterderivatives.

SUMMARY OF THE INVENTION

It has now been found that some17α,21-dihydroxypregna-4,9-diene-3,20-dione and17α,21-dihydroxypregna-4-ene-3,20-dione 17 and/or 21 esters haveremarkable antiandrogenic activity.

Therefore, according to a first embodiment, the present inventionrelates to compounds of formula (I)

wherein:R₁ and R₂, which can be the same or different, are hydrogen or a C₃-C₁₈acyl group, with the provisos that:

at least one of R₁ and R₂ is different from hydrogen;

when R₁ is hydrogen, R₂ is different from butyroyl.

According to a second embodiment, the invention relates to compounds offormula (II)

wherein:

R₁ and R₂, which can be the same or different, are hydrogen or a C₃-C₁₈acyl group, with the proviso that:

at least one of R₁ and R₂ is different from hydrogen; as antiandrogenicdrugs.

According to a further embodiment, the invention relates to a processfor the preparation of compounds of formula (I) or (II) in which R₁ andR₂ are both acyl, groups, which process comprises reacting thecorresponding compounds, wherein R₁ and R₂ are hydrogen, with carboxylicacids anhydrides or active esters in inert solvents and at temperaturesranging from −5° C. to the reaction mixture boiling temperature.

Still a further object of the invention relates to a process for thepreparation of compounds of formula (I) or (II) wherein one of R₁ or R₂is hydrogen and the other is acyl, which process comprises:

-   -   a. reaction of the corresponding compounds wherein R₁ and R₂ are        hydrogen with C₃-C₁₈ carboxylic acids anhydrides or active        esters or with allyloxycarbonyl chloride or isobutene in inert        solvents and at temperatures ranging from −5° C. to the boiling        temperature, for obtaining the corresponding compound in which        R₁ is isobutyl, allyloxycarbonyl or C₃-C₁₈ acyl;    -   b. optional reaction of the compound from step a) with C₃-C₁₈        carboxylic acids anhydrides or active esters in inert solvents        and at temperatures ranging from −5° C. to the reaction mixture        boiling temperature;    -   c. optional lysis of the 21-allyloxycarbonyl or 21-isobutyloxy        group.

Finally, the invention relates to pharmaceutical compositions withantiandrogenic activity containing as active ingredient the compounds offormula (I) or (II).

DETAILED DISCLOSURE OF THE INVENTION

Preferred compounds of formula (I) are:

-   -   17α,21-dibutanoyl-pregna-4,9-diene-3,20-dione;    -   17α-hydroxy-2′-butanoyl-pregna-4,9-diene-3,20-dione;    -   17α-hydroxy-21-butanoyl-pregna-49-di-ene-3,20-dione;    -   17α-butanoyl-2′-octadecanoyl-pregna-4,9-diene-3,20-dione;    -   17α-octadecanoyl-2′-butanoyl-pregna-4,9-diene-3,20-dione.

The antiandrogenic activity of the compounds of formula (I) and (II) hasbeen evaluated in the animal according to the conventional test for thetopical antiandrogenic activity described by W. Voigt and S. L. Hsia(Endocrinology 1973; 92: 1216-1222).

The test was carried out on sexually immature female hamsters aged 6-8weeks and weighing 65-90 grams.

At the beginning of the tests, the back of each animal was shaved toevidence the respective flank organ bilaterally. Animals were thensubdivided into homogeneous groups and treated daily for 21 consecutivedays. The tested steroids were dissolved at concentrations ranging from100 to 400 micrograms in 50 microlitres of an acetone solutioncontaining 4 micrograms of testosterone propionate (TP) or 4 microgramsof dihydrotestosterone (DHT). 50 Microlitres of the solutions wereapplied to the right flank organ, while the contralateral organ used asindividual control received only acetone (50 microlitres). Controlgroups received TP or DHT alone, following the same procedures.

At the end of the tests, the animals were killed under ether anesthesiaand the whole skine of the back was taken. The area of both flank organswas measured, separately, with transillumination. The mean differencesbetween areas treated with the tested steroids and those treated withthe carrier alone were calculated for each group, and said meandifferences were compared, as inhibition percentages, to the meandifferences between the areas in the control groups treated with eitherTP or DHT.

By way of example, in the topical antiandrogenic activity test17α-propionyl-21-hydroxy-pregna-4,9-diene-3,20-dione, and thecorresponding 17α,21-dibutyrate and 17α-butyrate inhibited by more than80% the androgenic action of testosterone propionate (TP) and by 50 to80% the action of its active derivative dihydrotestosterone (DHT).

The compounds of the invention proved active at doses ranging from 10 to4000 micrograms.

The compounds of the invention can be used as suitable pharmaceuticalcompositions for the topical and/or systemic treatment, through theoral, cutaneous or mucosal route, of conditions such as: acne,seborrhea, hirsutism, alopecia, mastodynia, prostate hyperplasia andcarcinoma, virilization syndromes in the female, early puberty,inhibition of sexual aggressiveness in the male, contraception in themale.

According to the process of the invention, compounds of formula (I) or(II) wherein R₁ and R₂ are both acyl groups are prepared byesterification of 17α,21-dihydroxypregna-4-ene-3,20-dione or17α,2′-dihydroxypregna-4,9-diene-3,20-dione hydroxy groups with activeesters containing the desired acyl group. According to this simpleprocedure, acyl derivatives with hindering aliphatic chains, such asthose with high number of carbon atoms or branched, can be prepared.Examples of suitable active esters for this reaction are trifluoroethylbutyrate or trifluoroethyl octadecanoate, which can both attainexcellent esterification yields with the aid of a lipase in inertanhydrous solvents at temperatures ranging from 20 to 50° C. and withreaction times ranging from 20 to 100 hours. Examples of lipases are PPL(porcine pancreatic lipase) or those from Candida cylindracea.

The process for the preparation of the compounds of formula (I) or (II)wherein one of R₁ o R₂ is hydrogen and the other is acyl comprises thefollowing steps:

1. The 21 hydroxyl is selectively esterified with allyloxycarbonylchloride, benzyloxy carbonyl chloride, tert-butylcarbonyl chloride indimethylformamide or isobutene at temperatures from −5 to 40° C.

2. The resulting 21 monoester is then subjected to esterification withanhydrides of carboxylic acids of 7 carbon atoms in the presence of4-dimethylaminopyridine as catalyst. Alternative to the esterificationin 17 is the use of the carboxylic acid in the presence ofdicyclohexylcarbodiimide. Active esters such as trifluoroethylderivatives or N-acylphthalimide or N-acylbenzotriazoles are furtheralternatives.

3. The protection in 21 is removed with, for example,tetrakis(triphenylphosphine) Pd and triphenyl phosphine indichloromethane or tetrahydrofuran to obtain17α-acyl-21-hydroxypregna-4-ene-3,20-dione or17α-acyl-21-hydroxypregna-4,9-diene-3,20-dione.

4. The product from step 3) can subsequently be esterified in 21 withanhydrides of carboxylic acids of 7 carbon atoms or alternatively withthe carboxylic acid in the presence of dicyclohexylcarbodiimide, or withactive esters such as trifluoroethyl derivatives or N-acylphthalimidesor N-acylbenzotriazoles.

Example 1 Preparation of 17α,21-dibutanoyl-pregna-4,9-diene-3,20-dione

A mixture of 1 g (2.87 mM) of17α,21-dihydroxy-pregna-4,9-diene-3,20-dione and of 10 ml oftrifluoroethyl butanoate in 50 ml of tetrahydrofuran was reacted at 45°C. in the presence of 5 g of Candida cylindracea lipase for 8-10 hours,adding 1 g of lipase at regular time intervals. At the end of this firstreaction step, the lipase was filtered off and the filtrate wasconcentrated under vacuum taking up the residue three times withtetrahydrofuran. The resulting residue was added with further 10 ml oftrifluoroethyl butanoate and 50 ml of tetrahydrofuran, the resultingsolution was added with 0.8 g of Bacillus subtilis protease and thesuspension was stirred for 2 days at 45° C., adding further protease atregular time intervals for total 80 mg. The protease was filtered off,the filtrate was removed under vacuum and the residue waschromatographed on a silica gel column with a dichloromethane/methanol99:1 mixture. The less polar fraction was evaporated to obtain 1 g (2.06mM) of 17α,21-dibutanoyl-pregna-4,9-diene-3,20-dione.

The same procedure was followed, starting from 1 g of17α,21-dihydroxy-pregna-4-ene-3,20-dione, to obtain 0.98 g (2.01 mM) of17α,21-dibutanoyl-pregna-4-ene-3,20-dione.

Example 2 Preparation of17α-hydroxy-21-butanoyl-pregna-4,9-diene-3,20-dione

A mixture of 1 g (2.879 mM) of17α,21-dihydroxy-pregna-4,9-diene-3,20-dione and 10 ml of trifluoroethylbutanoate in 100 ml of acetone was reacted at 45° C. in the presence of5 g of Candida cylindracea lipase for 8-10 hours, adding 1 g of lipaseat regular time intervals. After completion of the reaction, the lipasewas filtered off and the filtrate was concentrated under vacuum, takingup the residue three times with acetone. The semi-solid residue waspurified by chromatography on a silica gel column with adichloromethane/methanol 99:1 mixture. The less polar components wereremoved, to obtain the richer fraction which was evaporated to yield0.95 g (2.29 mM) of 17α-hydroxy-21-butanoyl-pregna-4,9-diene-3,20-dione.

Example 3 Preparation of 17α-hydroxy-21-butanoyl-pregna-4-ene-3,20-dione

A mixture of 1 g of 17α,21-dihydroxy-pregna-4-ene-3,20-dione and 10 mlof trifluoroethyl butanoate in 50 ml of methyl ethyl ketone was reactedat 45° C. in the presence of 5 g of Candida cylindracea lipase for 8-10hours, adding at regular time intervals 1 g of lipase. After completionof the reaction, the lipase was filtered off, the filtrate wasconcentrated under vacuum, taking up the residue three times withsolvent. The semi-solid residue was purified by chromatography on asilica gel column with a dichloromethane/methanol 99:1 mixture. Thericher fraction was evaporated to obtain 0.89 g (2.14 mM) of17α-hydroxy-21-butanoyl-pregna-4-ene-3,20-dione.

Example 4 Preparation of17α-butanoyl-21-octadecanoyl-pregna-4,9-diene-3,20-dione

4 g (11.6 mM) of 17α,21-dihydroxy-pregna-4,9-diene-3,20-dione werereacted with 20 mg of trifluoroacetic acid in 20 ml of dioxane and 10 mlof ethyl orthobutyrate for 5 hours at 100° C., and the low boiling headfraction was distilled off. The solution was cooled, then treated with 5ml of a tartaric acid molar solution and heated to 40-50° C. for about 5minutes to obtain 17α-butanoyl-21-hydroxy-pregna-4,9-diene-3,20-dione.The solvent was evaporated off under vacuum and the residue wasrepeatedly taken up with dioxane. The resulting residue was dissolved in200 ml of acetone and then 12 g trifluoroethyl octadecanoate (preparedfrom octadecanoyl chloride and trifluoroethanol), 20 g of Candidacylindracea lipase were added and the resulting suspension was stirredfor 8-10 hours at 50° C., adding 2 g of C. cylindracea at regular timeintervals. The lipase was filtered off, the filtrate was concentratedunder vacuum and the residue was chromatographed on a silica gel columnwith a dichloromethane/methanol 98.5:1.5 mixture. The neat fraction wasevaporated to obtain 4.9 g (7.17 mM) of17α-butanoyl-21-octadecanoyl-pregna-4,9-diene-3,20-dione.

The same procedure was followed, starting from 5 g (14.5 mM) of17α,21-dihydroxy-pregna-4-ene-3,20-dione, to obtain 5.9 g (8.61 mM) of17α-butanoyl-21-octadecanoyl-pregna-4-ene-3,20-dione.

Example 5 Preparation of17α-octadecanoyl-21-butanoyl-pregna-4,9-diene-3,20-dione

Step a: A solution of 2 g of NaOH in 20 ml of water was added with 25 mlof tetrahydrofuran and 5 g (14.5 mM) of17α,21-dihydroxy-pregna-4,9-diene-3,20-dione. The mixture was stirred at0° C., then 2.4 ml of allyl chloroformate was dropwise added. Afterstirring for about 0.5 hours at this temperature, the mixture wascarefully neutralized with hydrochloric acid and extracted withdichloromethane. The organic extract was concentrated under vacuum andthe residue was subjected to the reaction of the subsequent step.

Step b: Crude17α-hydroxy-21-allylcarbonyloxy-pregna-4,9-diene-3,20-dione wasdissolved in 15 g of trifluoroethyl octadecanoate and 150 ml oftetrahydrofuran, the resulting solution was added with 4 g of Bacillussubtilis protease and the suspension was stirred for 2 days at 45° C.,adding further protease at regular time intervals to 3 g total. Theprotease was filtered off, the filtrate was removed under vacuum and theresidue was chromatographed on a silica gel column with adichloromethane/methanol 99:1 mixture. The less polar fraction wasevaporated off to obtain a residue of17α-octadecanoyl-21-allylcarbonyloxy-pregna-4,9-diene-3,20-dione.

Step c: the residue from the previous step was dissolved in 50 ml ofdichloromethane and added with 35 mg of triphenylphosphine and 35 mg ofpalladium triphenylphosphine. The resulting mixture was stirred for 0.5hours at room temperature. The solution was concentrated under vacuum,the residue was taken up twice with dichloromethane, thenchromatographed on a silica gel column with a dichloromethane/methanol99:1 mixture. The richer fraction was evaporated to obtain a neatresidue, which was used as such for the subsequent step.

Step d: the residue (6.2 g) of17α-octadecanoyl-21-hydroxy-pregna-4-ene-3,20-dione was dissolved in 4ml butyric anhydride in the presence of 0.5 g of tributylmethylammoniumchloride. The mixture was stirred at room temperature for 2 hours, thenpoured in ice and the resulting product was separated from water byextraction. The extract was washed to neutrality with water andconcentrated under vacuum, the residue was crystallized from methanol toobtain 5.5 g (8.05 mM) of17α-octadecanoyl-21-butanoyl-pregna-4,9-diene-3,20-dione. This compoundwas used for the preparation of a pharmaceutical formulation in the formof a cream suitable for cutaneous administration.

The same procedure was followed, starting from 5 g (14.5 mM) of17α,21-dihydroxy-pregna-4-ene-3,20-dione, to obtain 5.1 g (7.44 mM) of17α-octadecanoyl-21-butanoyl-pregna-4-ene-3,20-dione.

The compounds of Examples 1-5 were formulated in suitable formulations,for example in the form of liposome emulsions or suspensions for thetransmucosal administration to provide either systemic or topicalaction, creams, gel and the like.

A typical cream formulation will contain, for example, cetyl alcohol,glycerol monostearate, liquid paraffin, propylene glycol, disodiummono-oleo-amido sulfosuccinate, citric acid monohydrate, purified water.

Using substantially the same methods disclosed in the above examples,the following compounds were prepared:

-   -   17α,21-dibutanoyl-pregna-4-ene-3,20-dione(mp 101° C., isopropyl        ether);    -   17α-propionyl-2′-hydroxy-pregna-4-ene-3,20-dione(mp 114° C.,        isopropyl ether).

Example 6 Topical Antiandrogenic Activity of17α-propionyl-21-hydroxy-pregna-4,9-diene-3,20-dione (Compound A)

Topical Mean difference of treatment Daily dosage (μg) the areas (mm²) %inhibition Carrier (acetone) — 0.0 — TP 4 22.7 ± 2.3  — TP + A 4 + 4003.7 ± 1.1 84 DHT 4 20.8 ± 2.5  — DHT + A 4 + 400 9.7 ± 1.8 53

Example 7 Topical Antiandrogenic Activity (Compound of Example 1)

Topical Mean difference of treatment Daily dosage (μg) the areas (mm²) %inhibition Carrier (acetone) — 0.0 — TP 4 22.7 ± 2.3  — TP + Ex. 1 4 +400 2.4 ± 1.1 89 DHT 4 20.8 ± 2.5  — DHT + Ex. 1 4 + 400 3.7 ± 0.7 82

Example 8 Topical Antiandrogenic Activity (Compound of Example 2)

Topical Mean difference of treatment Daily dosage (μg) the areas (mm²) %inhibition Carrier (acetone) — 0.0 — TP 4 22.7 ± 2.3  — TP + Ex. 2 4 +400 3.3 ± 1.2 85 DHT 4 20.8 ± 2.5  — DHT + Ex. 2 4 + 400 4.1 ± 0.5 80

1-8. (canceled)
 9. A method of treating a patient suffering from acne,seborrhea, hirsutism, alopecia, mastodynia, prostate hyperplasia andcarcinoma, virilization syndromes in the female, early puberty,inhibition of sexual aggressiveness in the male or contraception in themale, comprising administering to said patient topically or systemicallyan effective amount of a compound of formula (I)

wherein: R₁ is hydrogen and R₂ is a C₃-C₁₈ acyl group.
 10. A method oftreating a patient in need of antiandrogenic activity, said methodcomprising administering to said patient an effective amount of acompound of formula (II)

wherein: R₁ and R₂, which can be the same or different, are hydrogen ora C₃-C₁₈ acyl group, with the proviso that at least one of R₁ and R₂ isdifferent from hydrogen.
 11. The method as claimed in claim 10 whereinR₁ is hydrogen and R₂ is propionyl.
 12. The method as claimed in claim10 wherein R₁ and R₂ are butanoyl.
 13. A method of treating a patient inneed of antiandrogenic activity comprising administering to said patientan effective amount of a compound of formula (I) as defined in claim 9.14. A method of treating a patient suffering from acne, seborrhea,hirsutism, alopecia, mastodynia, prostate hyperplasia and carcinoma,virilization syndromes in the female, early puberty, inhibition ofsexual aggressiveness in the male or contraception in the male,comprising administering to said patient topically or systemically aneffective amount of a pharmaceutical composition comprising as activeingredient a compound of formula (I)

wherein: R₁ is hydrogen and R₂ is a C₃-C₁₈ acyl group, in admixture withan acceptable carrier, wherein said active ingredient is present in anamount of 0.1 to 1.0% by weight.
 15. The method as claimed in claim 13,wherein the compound of formula (I) is administered topically orsystemically, for the treatment of acne, seborrhea, hirsutism, alopecia,mastodynia, prostate hyperplasia and carcinoma, virilization syndromesin the female, early puberty, or contraception in the male.
 16. Themethod as claimed in claim 10, wherein the compound of formula (II) isadministered topically or systemically, for the treatment of acne,seborrhea, hirsutism, alopecia, mastodynia, prostate hyperplasia andcarcinoma, virilization syndromes in the female, early puberty, orcontraception in the male.
 17. A method as claimed in claim 9 whereinthe compound of formula (I) is as defined in claim 9, wherein R₁ ishydrogen and R₂ is propionate.
 18. A method as claimed in claim 9wherein the compound of formula (I) is as defined in claim 9, wherein R₁is hydrogen and R₂ is butyrate.
 19. A method as claimed in claim 14wherein the active ingredient is present in an amount of 0.2-0.8% byweight.